Regulator of G protein Signaling (RGS) proteins downregulate signaling through heterotrimeric G proteins by acting as G protein GTPase activators. They thus regulate signaling by serotonin, dopamine, and other neurotransmitters critical to human mental health. Some of the most important questions about RGS proteins remain unanswered. How are RGS proteins controlled so they can downregulate neural signaling only under appropriate circumstances? What do the various domains and subunits of RGS proteins do? This proposal seeks to answer these questions by carrying out a detailed analysis of two C. elegans RGS proteins, EGL-10 and EAT-16. These are so-called R7 RGS proteins, which have a domain similar to G protein 3 subunits and are constitutively bound to a divergent G2-like protein called G25. Genetic experiments in C. elegans strongly support the hypothesis that G25/RGS dimers function like the 23 component of conventional G123 heterotrimers, and thus assemble under certain circumstances with G1 proteins to form G1/25/RGS "unconventional heterotrimers". The unconventional heterotrimer model can explain 10 years of genetic results with EGL-10 and EAT-16 and provides a framework for understanding the whole purpose for which R7 RGS proteins exist. The proposed experiments will test the unconventional heterotrimer model using three independent lines experiments to analyze properties of the protein complexes formed by EGL-10 and EAT-16. The first aim will use co-immunoprecipitation experiments to test whether unconventional heterotrimers exist in C. elegans protein lysates, and to examine the properties of these protein complexes. A novel approach for rapidly generating biochemical quantities of lysates from transgenic C. elegans will be used to facilitate these experiments. The second aim will use both biochemical and genetic experiments to analyze the effects of an unusual mutant of G25 predicted to specifically disrupt unconventional heterotrimers. The third aim seeks to generate purified recombinant unconventional heterotrimer complexes and to analyze their biochemical properties. PUBLIC HEALTH RELEVANCE: Misregulation of signaling in the brain by neurotransmitters such as serotonin and dopamine leads to disorders such as depression and schizophrenia. This proposal seeks to understand the mechanisms that normally control levels of signaling by these neurotransmitters. This may lead to understanding the causes of human mental disorders and to therapies for their treatment.